CA1320461C - Monoclonal antibody capable of recognizing human arteriosclerosis and process for preparing same - Google Patents
Monoclonal antibody capable of recognizing human arteriosclerosis and process for preparing sameInfo
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Abstract
MONOCLONAL ANTIBODY CAPABLE OF
RECOGNIZING HUMAN ARTERIOSCLEROSIS AND
PROCESS FOR PREPARING THE SAME
BABSTRACT OF THE DISCLOSURE:
A monoclonal antibody capable of specifically recog-nizing a human arteriosclerosis related antigen is disclosed.
A process for preparing this monoclonal antibody using sera from arteriosclerotic patients or sites of arteriosclerotic lesions as antigens and a reagent for performing diagnosis of human arteriosclerosis using this antibody are also disclosed. The monoclonal antibody has the potential to be used not only as an index material in blood for direct diagnosis of human arteriosclerosis but also as an index material that directly recognizes arteriosclerotic lesions.
RECOGNIZING HUMAN ARTERIOSCLEROSIS AND
PROCESS FOR PREPARING THE SAME
BABSTRACT OF THE DISCLOSURE:
A monoclonal antibody capable of specifically recog-nizing a human arteriosclerosis related antigen is disclosed.
A process for preparing this monoclonal antibody using sera from arteriosclerotic patients or sites of arteriosclerotic lesions as antigens and a reagent for performing diagnosis of human arteriosclerosis using this antibody are also disclosed. The monoclonal antibody has the potential to be used not only as an index material in blood for direct diagnosis of human arteriosclerosis but also as an index material that directly recognizes arteriosclerotic lesions.
Description
MONOCLONAL ANTIBODY CAPABLE OF
RECOGNIZING HUMAN ARTERIOSCLEROSIS AND
PROCESS FOR PREPARING THE SAME
BACKGROUND OF THE INVENTION:
Field of the Invention The present invention relates to a human arterio-sclerosis recognizing monoclonal antibody that specifically recognizes a human arteriosclerosis related antigen. The present invention also relates to a process for preparing such monoclonal antibody. More particularly, the present invention provides a monoclonal antibody capable of recogniz-ing a human arteriosclerosis related antigen that is useful in such applications as the determination of a human arterio-sclerosis related antigen and diagnosis of arteriosclerosis based thereon, as well as imaging diagnosis of human arterio-sclerotic lesions.Description of the Prior Art Arteriosclerosis is a localized disease developed principally in large or middle sized arterias such as the aorta, coronary artery and cerebral artery and is a major cause of various occlusive diseases such as angina pectoris, myocardial infarction and cerebral infarction.
Many phenomena have so far been proposed as causes of arteriosclerosis and they include increased plasma choles-terol levels, damage to the endothelium, aggregation of blood platelets, swelling of the tunica intima and the formation of atheroma. However, little analysis has been made of the exact mechanism of these phenomena.
The normal aorta has a three-layered structure composed of an endothelium, a tunica media made of elastic tissues and smooth muscle cells, and a tunica externa made of elastic tissues. If, for some reason, the boundary between the endothelium and the tunica media swells and abnormal cell proliferation and necrosis occur, a disease will develop which is generally referred to as arteriosclerosis.
Among the principal causes of serious diseases such as myocardial infarction and cerebral infarction are:
(1) formation of atheroma due to the accumulation of cholesterol and other lipids in and between arterial wall cells;
RECOGNIZING HUMAN ARTERIOSCLEROSIS AND
PROCESS FOR PREPARING THE SAME
BACKGROUND OF THE INVENTION:
Field of the Invention The present invention relates to a human arterio-sclerosis recognizing monoclonal antibody that specifically recognizes a human arteriosclerosis related antigen. The present invention also relates to a process for preparing such monoclonal antibody. More particularly, the present invention provides a monoclonal antibody capable of recogniz-ing a human arteriosclerosis related antigen that is useful in such applications as the determination of a human arterio-sclerosis related antigen and diagnosis of arteriosclerosis based thereon, as well as imaging diagnosis of human arterio-sclerotic lesions.Description of the Prior Art Arteriosclerosis is a localized disease developed principally in large or middle sized arterias such as the aorta, coronary artery and cerebral artery and is a major cause of various occlusive diseases such as angina pectoris, myocardial infarction and cerebral infarction.
Many phenomena have so far been proposed as causes of arteriosclerosis and they include increased plasma choles-terol levels, damage to the endothelium, aggregation of blood platelets, swelling of the tunica intima and the formation of atheroma. However, little analysis has been made of the exact mechanism of these phenomena.
The normal aorta has a three-layered structure composed of an endothelium, a tunica media made of elastic tissues and smooth muscle cells, and a tunica externa made of elastic tissues. If, for some reason, the boundary between the endothelium and the tunica media swells and abnormal cell proliferation and necrosis occur, a disease will develop which is generally referred to as arteriosclerosis.
Among the principal causes of serious diseases such as myocardial infarction and cerebral infarction are:
(1) formation of atheroma due to the accumulation of cholesterol and other lipids in and between arterial wall cells;
(2) swelling of the tunica intima as a result of abnormal cell proliferation; and (3) aggregation of blood platelets as a result of damage to the endothelium and swelling of the tunica intima.
Because of these reasons, arterial vessels are occluded, presumably causing the development of such diseases as angina pectoris, myocardial infarction and cerebral infarction.
The following phenomena are typically found to have occurred in the swelling portion of the tunica intima:
(a) the appearance of foam cells taking up a large amount of lipids therein;
(b) accumulation of lipids between cells;
(c) proliferation of smooth muscle cells in the tunica intima;
(d) increased formation of connective tissues and the deposition of calcium; and (e) aggregation of blood platelets and formation of thrombi.
Diagnosis of human arteriosclerosis can be made either directly or indirectly. Indirect methods involve predicting the degree of risk based on the measurement of cholesterol levels in blood, analysis of lipoprotein composition and search for coagulation factors. Among the direct methods are echography techniques in which the progress of arterio-sclerosis is estimated by measuring the velocity of sound 10 propagating through the arterial wall or by utilizing echoes reflected by ultrasonic vibrations from the arterial wall, and angiography techniques in which an angiogram of the artery obtained by in;ecting an imaging medium into the arterial vessel is directly examined.
The determination of risk factors in blood as effected by the indirect methods does not involve the measurement of direct causatiye factors of arteriosclerosis and hence is not considered to be a highly reliable method of diagnosis. The direct methods, whether they are echographic or angiographic, 20 involve the measurement of the degree of blood vessel's constriction due to arteriosclerosis and are not directed to measuring the progress of the disease itself. Furthermore, the angiographic technique has a potential hazard on account of the need to in~ect an imaging medium into arteries.
It is, therefore, desired to develop a method of diag-nosis that is convenient and which has high specificity for human arteriosclerosis. In fact, however, neither an index material in blood that can be used for direct diagnosis of arteriosclerosis nor an index material that is capable of direct recognition of arteriosclerotic lesions has been found.
SUMMARY OF THE INVENTION:
In order to solve the aforementioned problems of the prior art, the present inventors conducted extensive studies on factors that would act directly on human arteriosclerosis.
As a result, the present inventors, using sera from arterio-sclerotic patients or sites of arteriosclerotic lesions as antigens, isolated monoclonal antibody producing cells that would specifically recognize antigens related to human arteriosclerotic diseases such as familial hypercholesterol-emia, myocardial infarction and cerebral infarction, and successfully obtained from these cells a monoclonal antibody capable of recognizing human arteriosclerosis.
Therefore, in one aspect, the present invention relates to a human arteriosclerosis recognizing monoclonal antibody capable of specifically recognizing a human arterio-sclerosis related antigen.
In another aspect, the present invention relates to a process for preparing a human arteriosclerosis recognizing monoclonal antibody comprising the steps of immunizing a non-human mammal with a solution containing a human arterio-sclerosis related antigen, fusing myeloma cells with antibody producing lymphocytes in the mammal, isolating hybridomas capable of producing an anti-human arteriosclerosis antibody, and cultivating said hybridomas.
In an advantageous convenient method, the monoclonal antibody of the present invention can be produced from hybridomas prepared by fusing anti-human arteriosclerosis antibody producing cells with myeloma cells. This method of production is in no way limited and may start with sensitiza-tion of a non-human mammal by standard techniques using a human arteriosclerosis antigen. Illustrative human arterio-sclerosis antigens include sera isolated from patients with human arteriosclerotic diseases such as familial hypercholes-terolemia, cerebral infarction and myocardial infarction, and a homogenate of arteriosclerotic lesion sites (in particular, a swelling portion of the tunica intima). When the sera are used as the antigens, they may be preferably mixed with anti-normal plasma anti-sera as illustrated in Example 1 hereunder whereby the subsequent sensitization can be efficiently conducted. In the next step of the method, lymphocytes capable of producing an anti-human arteriosclerosis antibody are isolated from the spleen of the sensitized animal, especially, the thymus, peripheral lymph nodes or peripheral blood, thereby obtaining cells capable of producing an anti-human arteriosclerosis antibody. These cells are then fused with myeloma cells by standard techniques to obtain antibody producing hybridomas. The hybridomas are distributed among a plurality of wells and cultivated. The supernatant of the culture in each well is assayed by a suitable technique such as enzyme immunoassay (ELISA) or indirect fluorescent antibody technique, so as to isolate human arteriosclerosis recognizing monoclonal antibody producing cells that bind specifically to the serum of a human arteriosclerotic patient or a human arteriosclerotic lesion and which does not t 32046 1 recognize the normal human serum or normal arterial wall.
The isolated cells are directly subjected to tissue cultivation. Alternatively, they may be transplanted in the abnormal cavity of a mammal, say, a mouse or guinea pig, to produce a tumor. The desired monoclonal antibody is collected from the ascites and purified.
The present inventors thus succeeded in isolating novel human arteriosclerosis recognizing monoclonal anti-bodies that specifically recognize human arteriosclerosis related antigens.
Among the monoclonal antibodies obtained in the present invention, monoclonal antibody 131B and others that were produced using sera from patients with hypercholes-terolemia, cerebral infarction and myocardial infarction as antigens are capable of specifically recognizing the arterio-sclerosis related antigens found in sera from human patients and are classified as IgG.
Monoclonal antibodies 125H and T256C could be obtained using as antigens the homogenates of swelling portions of the tunica intima of arteriosclerotic patients. Monoclonal anti-body 125H is classified as IgG and T256C as IgM; both were found to be capable of specifically recognizing arterio-sclerotic lesions.
BRIEF DESCRIPTION OF THE DRAWINGS:
Flg. 1 is a photograph showing the results of immuno-globulin classification of monoclonal antibody 131B by the Ouchterlony method;
Fig. 2 is a chart plotting the antigenic activities by disease of monoclonal antibody 131B based on the data shown in Table l;
Fig. 3 is a chart showing the results of protein and immunofluorescent staining of arteriosclerosis related substances recognized by monoclonal antibody 131B;
Fig. 4 is a graph showing the specific gravities of antigenic substances recognized by monoclonal antibody 131B;
Fig. 5 is a graph showing the corelationship between antigenic substances recognized by monoclonal antibody 131 and cholesterol levels in familial hypercholesterolemic patients;
Fig. 6 is a chart plotting the antigenic activities by disease of mDnoclonal antibody 125H based on the data shown in Table 2;
Fig. 7 is a micrograph showing the results of immuno-fluorescent staining by the indirect antibody technique of a frozen section of an arteriosclerotic lesion in rabbit that was allowed to react with monoclonal antibody 125H;
Fig. 8 is a micrograph showing the results of staining with Oil-Red O of a frozen section of an arteriosclerotic ; lesion in rabbit that was allowed to react with monoclonal antibody 125H;
Fig. 9 is a photograph showing the state of reaction between an arteriosclerotic lesion in rabbit and monoclonal antibody 125H as observed by indirect autoradiography;
Fig. 10 is a micrograph showing the state of the arteriosclerotic lesion that was allowed to react with monoclonal antibody 125H and which was observed by indirect autoradiography;
Fig. 11 is a photograph showing the results of protein and immunofluorescent staining of antigenic substances recognized by monoclonal antibody 125H;
Fig. 12 is a micrograph showing the results of immuno-fluorescent staining by the indirect fluorescent antibodytechnique of a section of a human arteriosclerotic lesion that was allowed to react with monoclonal antibody T256C;
Fig. 13 is a micrograph showing the results of stain-ing with Oil-Red O of a section of the same human arterio-sclerotic lesion that was allowed to react with mono~lonalantibody T256C, Fig. 14 is a micrograph showing the results of immuno-fluorescent ætaining by the indirect fluorescent antibody technique of a section of a rabbit arteriosclerotic lesion that was allowed to react with monoclonal antibody T256C; and Fig. 15 is a micrograph showing the results of stain-ing with Oil-Red O of a section of the same rabbit arterio-sclerotic lesion that was allowed to react with monoclonal antibody T256C.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS:
The monoclonal antibodies 131B, 125H and T256C were obtained by sensitization using sera from human patients or homogenates of the arterial wall as antigens and all of them were novel in that they differed from the monoclonal anti-bodies prepared by using rabbit sera and homogenates of thearterial wall as antigens.
The monoclonal antibodies of the present invention may be immediately used as reagents for estimating the progress of human arteriosclerosis. If deslred, these antibodies may be treated with proteolytic enzymes and the resulting decom-position products, F(ab')2 and Fab, used as reagents for estimating the progress of human arteriosclerosis. It is interesting to note that the antigens recognized by these monoclonal antibodies occur not only in human arterio-sclerotic lesion sites but also in sera from arteriosclerotic patients. Therefore, quantitative or qualitative analyses of the antigens present in sampled sera can be performed, using these monoclonal antibodies as reaction reagents, by a variety of known techniques including immunoassay techniques (e.g. competitive and sandwich methods) using markers such as enzymes (e.g. alkaline phosphatase, ~-galactosidase and peroxidase), radioisotopes and fluorescent materials, by measurements depending on agglutination reaction or reaction for inhibiting agglutinatlon, or by modifications of these methods. The results of these analyses can be used to estimate the progress of arteriosclerosis of interest.
The monoclonal antibodies of the present invention can also be used to detect the presence of a human arterio-sclerotic lesion site or to check the extent of its spread.
For instance, using radioisotopes such as Nal3lI, Na'23I and Nal2 5 I, iodine is bound to these monoclonal antibodies or fragments thereof such as F(ab')2 and Fab by such methods as the chloramine-T method and enzyme method. Technetium may be bound by adding a physiological saline solution of Na99mTcO~
(sodium pertechnetate) in the presence of a suitable reducing agent such as SnCl2. Alternatively, lndium (IllIn) may be bound with the aid of a suitable chelatlng agent [so-called bifunctional chelate such as diethylenetriamine pentaacetic acid (DTPA) anhydride~. The monoclonal antibodies or fragments thereof to which iodine, technetium or indium has been bound are then added to a sterile nontoxic medium and injected into veins. After a certain period of time, the results of association of the monoclonal antibodies or fragments thereof with the arteriosclerotic lesion are examined with an imaging device such as a gamma-camera to obtain a scintigram based on the distribution of radioac-tivity in the patient's body. The scintigram can be used to detect the presence of lesions of human arteriosclerotic diseases such as cerebral infarction and myocardial infarc-tion or to check the extent of their spread.
15The following examples are provlded for the purpose of further illustrating the present invention but are in no wayto be taken as limiting.
Example 1 Monoclonal Antibody of Class IgG Prepared Using i Sera from Hyperlipidemic Patients as Antigens (1) Preparation of monoclonal antibodies A portion (20 yl) of blood plasma taken from a normal sub~ect was diluted 10 folds with 180 ~1 of physiological saline and administered intraperitoneally into BALB/c mice.
Following this in~ection, the same dilution was administered intraperitoneally after 3 weeks and 2 months, thereby obtain-ing anti-normal plasma anti-sera. Sera taken from 19 famil-ial hyperlipidemic patients were mixed together and 20 ~1 of the mixture was diluted 5 folds with 80 ~1 of physiological saline. The dilution was intimately mixed with 100 ~1 of a Freund's complete adjuvant and the resulting emulsion was administered subcutaneously to BALB/c mice. After 2 weeks, 10 ~1 of the previously prepared anti-normal plasma anti-sera and 10 ~1 of a mixed serum from 19 familial hyperlipidemic patients were diluted with 80 yl of physiological saline.
The dilution was intimately mixed with 100 ~1 of a Freund's complete ad~uvant and the resulting emulsion was administered subcutaneously into the animals. After 2 weeks, the mice were finally immunized by intraperitoneal injection of a dilution in physiological saline (180 ~1) of 10 ~1 of the anti-normal plasma anti-sera and 10 ~1 of a mixed sera of 19 familial hyperlipidemlc patients. On the third day, spleens were extracted from the sensitized animals.
The spleen cells were thoroughly washed with HHBS
(Hepes buffered Hanks' balanced salt solution), mixed with thoroughly washed mouse myeloma cells (strain P3/Ul) at a ratio of 5:1, and centrifuged at 1,300 rpm for 5 minutes.
The cell pellet was suspended in 1 ml of DMEM(-) medium containing 50% polyethylene glycol 4000 and left to stand for 2 minutes. Subsequently, 10 ml of DMEM(-) medium was slowly added to make a dilution, which was centrifuged at 800 rpm for 5 minutes. The cell pellet was resuspended in 100 ml of a HAT medium containing 20% fetal calf serum and the suspen-sion was distributed in O.l-ml portions among 96 wells in a tissue culture plate. Every 2 - 4 days, half the medium was replaced with a fresh medium. Investigation of antibody titer conducted on the supernatants of the culture after 8 days showed that strong antibody activities were found in 21 of 1,000 wells. Cloning was then performed by a limiting dilution technique so as to select clones that would not show any antibody activity against a mixed plasma of 14 normal subjects or a mixed serum of 14 normal subjects but which would specifically recognize arteriosclerosis related antigens in human sera. As a result, a total of 9 clones of hybridomas were obtained. The final clonins was conducted by a limiting dilution technique so as to isolate a total of 6 clones of hybridomas that were more inactive against normal plasma and sera but which specifically reacted with sera from patients suffering from arteriosclerosis related diseases.
These clones were in~ected intraperitoneally into pristan-treated BALB/c mice and after 10 - 20 days, ascites was colleeted from each animal to obtain monoelonal antibodies.
Of the monoelonal antibodies obtained from the 6 clones of hybridomas, those obtained from cell line 131B
were found to be of immunoglobulin subclass IgG~ by the Ouchterlony method (see Fig. 1) and they had the ability to specifically recognize arteriosclerosis related antigens in human sera. Cell strain 131B has been deposited under the Budapest Treaty with the Fermentation Researeh Institute (FERM) the Ageney of Industrial Seience and Technology, an international depository authority, under Accession Number FERM BP-1676.
(2) Assaying patient sera with monoclonal antibody 131B
The sera to be tested were as follows: serum from a familial hyperlipidemic patient (FH) serum from type III
hyperlipidemic patient (Type III); serum from hypo-beta-lipoproteinemic patient (Hypo ~); serum from patient with myocardial infarction (MI); serum from patient with cerebral infarction (APO); serum from patient with Werner's syndrome (Werner); serum from patient with angina pectoris (Angina);
plasma from normal sub~ect (NP); and a mixed serum prepared from blood samples of 14 normal subjects (NS). Each of these sera was diluted 1,000 folds and 100 ~1 of each dllution was added to an ELISA microplate (Falcon 3912). The plate was left to stand overnight at 4~ to have the homogenate ad-sorbed on it. After treating the microplate with a phosphate buffer solution containing 1% BSA, 100 ~1 of a solution of monoclonal antibody 131B was added and reaction was performed at 37C for 2 hours. After washing the microplate, 100 ~1 of 10,000-fold diluted alkaline phosphatase labelled anti-mouse IgG (Tago) was added and reaction was carried out at 37C for 1 hour. After washing the microplate, 100 ml of 1 mM
diethanolamine buffer solution (pH, 9.8) containing 1 mg/ml of disodium salt of paranitrophenylphosphoric acid and 0.01%
MgCl2 was added and reaction was carried out at 37C for 2 hours. Light absorbance values (OD3 D 5 nm) measured with a microplate colorimeter (Bio-Rad) were used as indices of activity against arteriosclerosis related antigens. The results are shown in Table 1. The monoclonal antibody 131B
of the present invention exhibited high activity levels through highly specific recognition of arteriosclerosis related antigens in FH, Type III, Hypo ~, MI, APO, Werner and Angina. In addition, this monoclonal antibody showed only low activity levels against NP and NS. It was, therefore, clear that monoclonal antibody 131B was capable of specifi-cally recognizing arteriosclerosis related antigens in human sera.
These results are shown graphically in Fig. 2, from which one can see that all sera from hypercholesterolemic patient, Type III hyperlipidemic patient, hypo-beta-lipoproteinemic patient, myocardial infarction patient and cerebral infarction patient showed higher antigenic activity levels than normal plasma and sera.
(3) Arteriosclerosis related antigenic substances in human serum capable of reacting with monoclonal antibody 131B
Sera from familial hypercholesterolemic patients were sub~ected to electrophoresis through an SDS-added polyacryl-amide gel overnight at 40 volts and blotted on a nitro-cellulose membrane. The membrane was thoroughly washed with a phosphate buffer solution containing 0.05% nonionic surfac-tant Tween 20 and thereafter treated overnight with a phos-phate buffer solution containing 5~ 8SA. After washing, the 20 membrane was sub~ected to reaction with monoclonal antibody 131B for 2 hours, then with alkaline phosphatase labelled anti-mouse IgG antibody for 1 hour. After the reaction, the membrane was immersed in a 0.75 M Tris-HCl buffer solution (pH, 8.8) containing 0.2~ paratoluidine salt of 5-bromo-4-chloro-3-indolylphosphoric acid and left to stand overnight at room temperature for immunofluorescent staining. It was estimated from the results of staining that the arterio-sclerosis related antigenic substances in human serum that were specifically recognized by monoclonal antibody 131B had molecular weights of approximately 52,000.
The antigenic substances recognized by monoclonal antibody 131B have specificity for hyperlipidemia and differ from lipoprotein constituents, such as Apo A-I, A-IV, B-100, B-48, E and D, which are said to be closely related to arteriosclerosis (see Fig. 3).
As shown in Fig. 4, the antigenic substances recog-nized by monoclonal antibody 131B were found in fractions that did not float even after 24-hour centrifugation at 30,000 rpm of 1 ml of serum from a familial hyperlipidemic patient that had been ad~usted to a specific gravity of 1.25 with NaBr and thereafter plated with 2 ml of physiological saline having a specific gravity of l.lS3 and 1.6 ml of physiological saline having a specific gravity of 1.063.
Furthermore, the low degree of correlation (R = 0.104) with the cholesterol level of familial hyperlipidemic patients showed that monoclonal antibody 131B does not recognize LDL
or lipoproteins as antigenic substances.
From these results, it is concluded that monoclonal antibody 131B specifically recognizes those antigenic substances which are characteristic of arteriosclerotic diseases and which are commonly found in patient's sera.
It has also been found that this monoclonal antibody is capable of specific recognition of antigenic substances in blood plasma, too.
Table 1 Cholesterol Levels of Sera from Patients Suffering from Arteriosclerosis Related Diseases and Their Antigenic Activities for Monoclonal Antibody 131B
~eas~ ~O~l- Patient No. (mg/dl) ELISA
FH 1 73-02615 91 0.551 FH 2 81-06627 159 0.372 FH . 3 80-48341 219 0.300 FH 4 85-12795 176 0.299 FH 5 78-01909 179 0.223 FH 6 81-42373 223 0.340 FH 7 82-59757 188 0.399 FH 8 82-60373 232 0.376 FH 9 83-11984 223 0.314 FH 10 85-18298 216 0.292 FH 11 81-42373 195 0.259 FH 12 82-57486 225 0.205 FH 13 78-03839 320 0.570 FH 14 78-00694 285 0.434 FH 15 86-26248 245 0.580 FH 16 81-58703 244 0.937 FH 17 86-13848 276 0.650 FH 18 85-46484 250 0.351 FH 19 81-14720 274 0.629 FH 20 78-03537 274 0.591 FH 21 78-04523 241 0.747 FH 22 79-66446 258 0.715 FH 23 79-07111 260 0.832 Table 1 (cont'd) DiseaseSamplePatient No.(mg/dl) ELISA
FH 24 80-19562 241 0.712 FH 25 78-07831 345 0.808 FH 26 83-35843 342 0.561 FH 27 70-01093 236 1.069 FH 28 87-30840 304 0.269 FH 29 80-05283 223 0.681 FH 30 86-18468 229 0.603 FH 31 86-26301 212 0.471 FH 32 85-37071 178 0.446 FH 33 82-29714 215 0.401 FH 34 80-41131 276 0.535 FH 35 80-06511 254 0.211 FH 36 82-45798 211 0.621 FH 37 78-03381 260 0.349 FH 38 87-19069 292 0.546 FH 39 86-33547 205 0.478 FH 40 85-19289 247 0.573 FH 41 87-23854 268 0.724 FH 42 85-04596 227 0.697 FH 43 86-19522 273 0.785 FH 44 79-64322 170 0.475 FH 45 84-29516 226 0.807 FH 46 85-18616 153 0.822 FH 47 81-37201 235 0.549 FH 48 79-36564 202 0.565 able 1 (cont'd) Disease ~Patieot No. (mg/dl) ELISA
FH 49 78-06522 225 0.756 FH 50 78-08511 209 0.291 FH 51 86-07503 199 0.546 FH 52 78-05045 278 0.622 FH 53 86-39926 250 0.586 FH 54 85-32090 162 0.558 FH 55 85-33074 115 0.521 FH 56 81-16704 190 0.727 FH 57 80-27988 251 0.737 FH 58 87-14349 290 0.689 FH 59 80-27368 207 0.818 : FH 60 86-01605 276 0.390 FH 61 84-45499 131 0.951 AP0 62 78-01164 106 0.247 AP0 63 83-18926 123 0.534 AP0 64 78-00900 115 0.939 AP0 65 73-00300 163 0.259 AP0 66 78-09966 148 0.186 AP0 67 78-03785 163 0.360 AP0 68 86-25083 187 0.282 AP0 69 78-00849 199 0.271 AP0 70 78-05165 185 0.296 AP0 7184-10579 205 0.496 AP0 7273-06841 117 0.451 AP0 7378-01373 131 0.302 AP0 7477-02200 170 0.291 Table 1 (cont'd) Di-e~seS~n~l e Patient No. (mg/dl) ELISA
MI 75 84-00571 164 0.335 MI 76 78-00485 161 0.499 MI 77 78-04516 133 0.433 MI 78 78-02674 177 0.423 MI 79 74-03633 178 0.483 MI 80 73-03652 105 0.823 Hypo ~ 81 82-21911 99 0.457 Hypo ~ 82 87-32118 91 0.506 Hypo ~ 83 78-03270 67 0.280 Hypo ~ 84 80-45830 93 0.346 Hypo ~ 85 84-26556 78 0.444 Hypo ~ 86 85-20771 114 0.372 Hypo ~ 87 83-13324 100 0.251 Hypo ~ 88 78-01437 101 0.517 Type III89 79-31979 198 0.545 Type III90 725900 243 0.520 Werner 100 85-40293 183 0.507 Werner 101 81-18040 147 0.447 Anglna 102 83-20213 182 0.205 T.C. : Total cholesterol FH : Familial hyperlipidemia AP0 : Cerebral infarction MI : Myocardial infarction Hypo ~ : Hypo-~-lipoproteinemia Type III : Hyperlipidemia type III
Werner : Werner's syndrome Angina : Angina pectoris Example 2 Arteriosclerotic Wall Recognizing Monoclonal Antibody of Class IgG Prepared Using Arteriosclerotic Lesions as Antigens (1~ Preparation of antigen Human thoracic and abdominal arteriosclerotic walls were extracted and blood vessels were excised in a cool place. Thereafter, the swelling portion of the tunica intima (arteriosclerotic lesion) was peeled off with tweezers and cut into squire pieces (1 mm x 1 mm) with scissors. To these pieces, an aqueous solution (pH, 7.~) containing 1 mM EDTA and 0.1% ethanol was added in portions of 5 - 10 ml per gram on a wet weight basis and a homogenate was prepared with a polytron homogenizer.
A mixture of homogenates originating from 3 patients (equlvalent to 5 specimens) was filtered through 4 superposed sheets of gauze and the filtrate was used as an antigenic solution.
(2) Preparation of monoclonal antibodies To 0.5 mg (200 ~1) of the homogenate, 200 ~1 of Freund's complete ad~avant was added and mixed well. The resulting emulsion was administered subcutaneously into BALB/c mice. After 9 weeks, the same emulsion was admin-istered subcutaneously. After 18 weeks, the final immuniza-tion was conducted by intraperitoneal in;ection of a mixture of 0.5 mg (200 ~1) of the homogenate with 200 ~1 of a DMEM(-) medium. On the ~hird day, spleens were extracted from the sensitized animals. The spleen cells were thoroughly washed with ~MEM(-) medium and mixed with 1 3204hl thoroughly washed mouse myeloma cells (strain P3/Ul) at a ratio of 5:1 and the mixture was centrifuged at 1,000 rpm for 5 minutes.
The cell pellet was suspended in 1 ml of DMEM( - ) medium containing 45% polyethylene glycol 4000 and left to stand for 2 minutes. Subsequently, 10 ml of DMEM( - ) medium was slowly added to make a dilution, which was centrifuged at 1,000 rpm for 5 minutes. The cell pellet was re-suspended in 127 ml of a DMEM medium containing 10% fetal calf serum and the suspension was distributed in l-ml portions among 24 wells in a tissue culture plate.
On the first day of culture, 1 ml of a HAT medium was added and half the medium was replaced with a fresh medium every 1 - 2 days. On the 8th day, the antibody titers of the culture supernatants were examined by the ELISA method.
The previously prepared homogenate of the swelling portion of the tunica intima of human arteriosclerotic lesion was diluted to make a solution having a protein concentration of 10 ~g/ml. A 100-~1 portion of this antigenic solution was added to an ELISA microplate (Nunc), which was left to stand overnight at 4C to have the homogenate adsorbed on it. After treating the microplate with a 2% BSA solution, 100 yl of the culture supernatants from 127 wells was added and reaction was conducted for 2 hours at room temperature. After washing the microplate, 100 ~1 of 6,000-fold diluted alkaline phosphatase labelled anti-mouse IgG antibody (Tago) was added and reaction was carried out for 2 hours at room temperature. After washing the microplate, 100 ~1 of a 1 mM diethanolamine buffer solution (pH, 9.8) containing 1 mg/ml of disodium salt of paranitrophenylphosphoric acid and 0.01% MgCl 2 was added and reaction was carried out for 1 hour at room temperature.
Light absorbance ~OD~os nm) was measured with a microplate colorimeter (Bio-Tech). Sixty-five wells having absorbance values of 0.5 and higher were further examined by autoradiography.
Arteriosclerotic lesions were extracted from arteriosclerotic model rabbits that had been grown with 1% cholesterol-supplemented diets for 4 months, and the extracted lesions were cut into 65 pieces. After treating these pieces with a 5~ BSA solution, 1 ml of the culture supernatants from the 65 wells was added and reaction was 15 performed at 4C for 5 hours. After washing, the reaction mixture was treated with 5~ normal sheep serum and 200 yl (1.7 Ci/ml) of 12 5 I labelled anti-mouse IgG antibody (unlabelled; Tago) was added, followed by reaction at 4C
for 3 hours. After washing, the reaction mixture was 20 subjected to autoradiography with X-ray films (FUJI*Photo Film).
From the 65 wells tested, 10 wells that produced a specific reaction and exhibited high binding efficiencies were selected and subjected to cloning by a limiting dilution technique. As a result, a total of 2 clones of hybridoma was isolated. These hybridomas were injected intraperitoneally into pristan-treated BALB/c mice and a~ter 10 - 16 days,-ascites was collected from each animal to * Trade mark Bl obtain monoclonal antibod,ies. Of the monoclonal antibodies obtained from the 2 clones of hybridomas, those obtained from cell line 125H were found to be immunoglobulin subclass I3G2b by the ELISA method and they had the ability to specifically recognize the swelling portion of the tunica intima of arteriosclerotic wall. Cell line 125H has been deposited under the Budapest Treaty with the FERM, an international depository authority, under Accession Number FERM B~-1675.
(3) Specificity of monoclonal antibody 125H
Using 100 yl of homogenates of the swelling portions of tunica intima of various human arteriosclerotic lesions and those of tunica intima of normal artery (each homogenate being adjusted to a protein content of 10 yg/ml), the reac-tivity of monoclonal antibody 125H was investigated by theELISA method. The results are shown in Table 2 and Fig. 6.
It was established that this monoclonal antibody shows strong activity with human arteriosclerotic lesions.
Measurements of total cholesterol (TC) levels were conducted with a cholesterol assay kit (V-cholestase NISSUI* of Nissui Seiyaku Co., Ltd.) The reactivity of monoclonal antibody 125H with the swelling portion of a rabbit arteriosclerotic tunica intima was also investigated. The results of immunofluorescent staining (by the indirect fluorescent antibody technique) conducted on frozen sections of the arteriosclerotic lesions in arteriosclerotic model rabbits that had been grown with 1% cholesterol-supplemented diets are shown in Fig. 7, and * Trade mark - 23 -the results of staining with Oil-Red O conducted on frozen sections of the same lesions are shown in Fig. 8.
Arteriosclerotic lesions were also extracted from another group of arteriosclerotic model rabbits that had 5 been grown with 1% cholesterol-supplemented diets and the reactivity of monoclonal antibody with these lesions was examined by autoradiography. The results of reaction by indirect autoradiography and the state of the lesions after the reaction are shown in Figs. 9 and 10, respectively.
10 As is clear from these figures, monoclonal antibody 125H
specifically recognized the swelling portion of rabbit arteriosclerotic tunica intima.
On the basis of these results, one may be ~ustified to expect that monoclonal antibody 125H will react with .
arteriosclerotic lesions after administration in vivo. When one wants to prepare a reagent for image analysis to be performed as an ad~unct in the diagnosis of human arterio-sclerosis, he is capable of conducting extensive preliminary tests on dosage, safety and other factors using rabbits before applying said reagent to humans. Therefore, mono-clonal antibody 125H has a potential application in image analyses fo~ the purpose of identifying the site of a specific human arteriosclerotic lesion and examining the extent of its spread.
Because of these reasons, arterial vessels are occluded, presumably causing the development of such diseases as angina pectoris, myocardial infarction and cerebral infarction.
The following phenomena are typically found to have occurred in the swelling portion of the tunica intima:
(a) the appearance of foam cells taking up a large amount of lipids therein;
(b) accumulation of lipids between cells;
(c) proliferation of smooth muscle cells in the tunica intima;
(d) increased formation of connective tissues and the deposition of calcium; and (e) aggregation of blood platelets and formation of thrombi.
Diagnosis of human arteriosclerosis can be made either directly or indirectly. Indirect methods involve predicting the degree of risk based on the measurement of cholesterol levels in blood, analysis of lipoprotein composition and search for coagulation factors. Among the direct methods are echography techniques in which the progress of arterio-sclerosis is estimated by measuring the velocity of sound 10 propagating through the arterial wall or by utilizing echoes reflected by ultrasonic vibrations from the arterial wall, and angiography techniques in which an angiogram of the artery obtained by in;ecting an imaging medium into the arterial vessel is directly examined.
The determination of risk factors in blood as effected by the indirect methods does not involve the measurement of direct causatiye factors of arteriosclerosis and hence is not considered to be a highly reliable method of diagnosis. The direct methods, whether they are echographic or angiographic, 20 involve the measurement of the degree of blood vessel's constriction due to arteriosclerosis and are not directed to measuring the progress of the disease itself. Furthermore, the angiographic technique has a potential hazard on account of the need to in~ect an imaging medium into arteries.
It is, therefore, desired to develop a method of diag-nosis that is convenient and which has high specificity for human arteriosclerosis. In fact, however, neither an index material in blood that can be used for direct diagnosis of arteriosclerosis nor an index material that is capable of direct recognition of arteriosclerotic lesions has been found.
SUMMARY OF THE INVENTION:
In order to solve the aforementioned problems of the prior art, the present inventors conducted extensive studies on factors that would act directly on human arteriosclerosis.
As a result, the present inventors, using sera from arterio-sclerotic patients or sites of arteriosclerotic lesions as antigens, isolated monoclonal antibody producing cells that would specifically recognize antigens related to human arteriosclerotic diseases such as familial hypercholesterol-emia, myocardial infarction and cerebral infarction, and successfully obtained from these cells a monoclonal antibody capable of recognizing human arteriosclerosis.
Therefore, in one aspect, the present invention relates to a human arteriosclerosis recognizing monoclonal antibody capable of specifically recognizing a human arterio-sclerosis related antigen.
In another aspect, the present invention relates to a process for preparing a human arteriosclerosis recognizing monoclonal antibody comprising the steps of immunizing a non-human mammal with a solution containing a human arterio-sclerosis related antigen, fusing myeloma cells with antibody producing lymphocytes in the mammal, isolating hybridomas capable of producing an anti-human arteriosclerosis antibody, and cultivating said hybridomas.
In an advantageous convenient method, the monoclonal antibody of the present invention can be produced from hybridomas prepared by fusing anti-human arteriosclerosis antibody producing cells with myeloma cells. This method of production is in no way limited and may start with sensitiza-tion of a non-human mammal by standard techniques using a human arteriosclerosis antigen. Illustrative human arterio-sclerosis antigens include sera isolated from patients with human arteriosclerotic diseases such as familial hypercholes-terolemia, cerebral infarction and myocardial infarction, and a homogenate of arteriosclerotic lesion sites (in particular, a swelling portion of the tunica intima). When the sera are used as the antigens, they may be preferably mixed with anti-normal plasma anti-sera as illustrated in Example 1 hereunder whereby the subsequent sensitization can be efficiently conducted. In the next step of the method, lymphocytes capable of producing an anti-human arteriosclerosis antibody are isolated from the spleen of the sensitized animal, especially, the thymus, peripheral lymph nodes or peripheral blood, thereby obtaining cells capable of producing an anti-human arteriosclerosis antibody. These cells are then fused with myeloma cells by standard techniques to obtain antibody producing hybridomas. The hybridomas are distributed among a plurality of wells and cultivated. The supernatant of the culture in each well is assayed by a suitable technique such as enzyme immunoassay (ELISA) or indirect fluorescent antibody technique, so as to isolate human arteriosclerosis recognizing monoclonal antibody producing cells that bind specifically to the serum of a human arteriosclerotic patient or a human arteriosclerotic lesion and which does not t 32046 1 recognize the normal human serum or normal arterial wall.
The isolated cells are directly subjected to tissue cultivation. Alternatively, they may be transplanted in the abnormal cavity of a mammal, say, a mouse or guinea pig, to produce a tumor. The desired monoclonal antibody is collected from the ascites and purified.
The present inventors thus succeeded in isolating novel human arteriosclerosis recognizing monoclonal anti-bodies that specifically recognize human arteriosclerosis related antigens.
Among the monoclonal antibodies obtained in the present invention, monoclonal antibody 131B and others that were produced using sera from patients with hypercholes-terolemia, cerebral infarction and myocardial infarction as antigens are capable of specifically recognizing the arterio-sclerosis related antigens found in sera from human patients and are classified as IgG.
Monoclonal antibodies 125H and T256C could be obtained using as antigens the homogenates of swelling portions of the tunica intima of arteriosclerotic patients. Monoclonal anti-body 125H is classified as IgG and T256C as IgM; both were found to be capable of specifically recognizing arterio-sclerotic lesions.
BRIEF DESCRIPTION OF THE DRAWINGS:
Flg. 1 is a photograph showing the results of immuno-globulin classification of monoclonal antibody 131B by the Ouchterlony method;
Fig. 2 is a chart plotting the antigenic activities by disease of monoclonal antibody 131B based on the data shown in Table l;
Fig. 3 is a chart showing the results of protein and immunofluorescent staining of arteriosclerosis related substances recognized by monoclonal antibody 131B;
Fig. 4 is a graph showing the specific gravities of antigenic substances recognized by monoclonal antibody 131B;
Fig. 5 is a graph showing the corelationship between antigenic substances recognized by monoclonal antibody 131 and cholesterol levels in familial hypercholesterolemic patients;
Fig. 6 is a chart plotting the antigenic activities by disease of mDnoclonal antibody 125H based on the data shown in Table 2;
Fig. 7 is a micrograph showing the results of immuno-fluorescent staining by the indirect antibody technique of a frozen section of an arteriosclerotic lesion in rabbit that was allowed to react with monoclonal antibody 125H;
Fig. 8 is a micrograph showing the results of staining with Oil-Red O of a frozen section of an arteriosclerotic ; lesion in rabbit that was allowed to react with monoclonal antibody 125H;
Fig. 9 is a photograph showing the state of reaction between an arteriosclerotic lesion in rabbit and monoclonal antibody 125H as observed by indirect autoradiography;
Fig. 10 is a micrograph showing the state of the arteriosclerotic lesion that was allowed to react with monoclonal antibody 125H and which was observed by indirect autoradiography;
Fig. 11 is a photograph showing the results of protein and immunofluorescent staining of antigenic substances recognized by monoclonal antibody 125H;
Fig. 12 is a micrograph showing the results of immuno-fluorescent staining by the indirect fluorescent antibodytechnique of a section of a human arteriosclerotic lesion that was allowed to react with monoclonal antibody T256C;
Fig. 13 is a micrograph showing the results of stain-ing with Oil-Red O of a section of the same human arterio-sclerotic lesion that was allowed to react with mono~lonalantibody T256C, Fig. 14 is a micrograph showing the results of immuno-fluorescent ætaining by the indirect fluorescent antibody technique of a section of a rabbit arteriosclerotic lesion that was allowed to react with monoclonal antibody T256C; and Fig. 15 is a micrograph showing the results of stain-ing with Oil-Red O of a section of the same rabbit arterio-sclerotic lesion that was allowed to react with monoclonal antibody T256C.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS:
The monoclonal antibodies 131B, 125H and T256C were obtained by sensitization using sera from human patients or homogenates of the arterial wall as antigens and all of them were novel in that they differed from the monoclonal anti-bodies prepared by using rabbit sera and homogenates of thearterial wall as antigens.
The monoclonal antibodies of the present invention may be immediately used as reagents for estimating the progress of human arteriosclerosis. If deslred, these antibodies may be treated with proteolytic enzymes and the resulting decom-position products, F(ab')2 and Fab, used as reagents for estimating the progress of human arteriosclerosis. It is interesting to note that the antigens recognized by these monoclonal antibodies occur not only in human arterio-sclerotic lesion sites but also in sera from arteriosclerotic patients. Therefore, quantitative or qualitative analyses of the antigens present in sampled sera can be performed, using these monoclonal antibodies as reaction reagents, by a variety of known techniques including immunoassay techniques (e.g. competitive and sandwich methods) using markers such as enzymes (e.g. alkaline phosphatase, ~-galactosidase and peroxidase), radioisotopes and fluorescent materials, by measurements depending on agglutination reaction or reaction for inhibiting agglutinatlon, or by modifications of these methods. The results of these analyses can be used to estimate the progress of arteriosclerosis of interest.
The monoclonal antibodies of the present invention can also be used to detect the presence of a human arterio-sclerotic lesion site or to check the extent of its spread.
For instance, using radioisotopes such as Nal3lI, Na'23I and Nal2 5 I, iodine is bound to these monoclonal antibodies or fragments thereof such as F(ab')2 and Fab by such methods as the chloramine-T method and enzyme method. Technetium may be bound by adding a physiological saline solution of Na99mTcO~
(sodium pertechnetate) in the presence of a suitable reducing agent such as SnCl2. Alternatively, lndium (IllIn) may be bound with the aid of a suitable chelatlng agent [so-called bifunctional chelate such as diethylenetriamine pentaacetic acid (DTPA) anhydride~. The monoclonal antibodies or fragments thereof to which iodine, technetium or indium has been bound are then added to a sterile nontoxic medium and injected into veins. After a certain period of time, the results of association of the monoclonal antibodies or fragments thereof with the arteriosclerotic lesion are examined with an imaging device such as a gamma-camera to obtain a scintigram based on the distribution of radioac-tivity in the patient's body. The scintigram can be used to detect the presence of lesions of human arteriosclerotic diseases such as cerebral infarction and myocardial infarc-tion or to check the extent of their spread.
15The following examples are provlded for the purpose of further illustrating the present invention but are in no wayto be taken as limiting.
Example 1 Monoclonal Antibody of Class IgG Prepared Using i Sera from Hyperlipidemic Patients as Antigens (1) Preparation of monoclonal antibodies A portion (20 yl) of blood plasma taken from a normal sub~ect was diluted 10 folds with 180 ~1 of physiological saline and administered intraperitoneally into BALB/c mice.
Following this in~ection, the same dilution was administered intraperitoneally after 3 weeks and 2 months, thereby obtain-ing anti-normal plasma anti-sera. Sera taken from 19 famil-ial hyperlipidemic patients were mixed together and 20 ~1 of the mixture was diluted 5 folds with 80 ~1 of physiological saline. The dilution was intimately mixed with 100 ~1 of a Freund's complete adjuvant and the resulting emulsion was administered subcutaneously to BALB/c mice. After 2 weeks, 10 ~1 of the previously prepared anti-normal plasma anti-sera and 10 ~1 of a mixed serum from 19 familial hyperlipidemic patients were diluted with 80 yl of physiological saline.
The dilution was intimately mixed with 100 ~1 of a Freund's complete ad~uvant and the resulting emulsion was administered subcutaneously into the animals. After 2 weeks, the mice were finally immunized by intraperitoneal injection of a dilution in physiological saline (180 ~1) of 10 ~1 of the anti-normal plasma anti-sera and 10 ~1 of a mixed sera of 19 familial hyperlipidemlc patients. On the third day, spleens were extracted from the sensitized animals.
The spleen cells were thoroughly washed with HHBS
(Hepes buffered Hanks' balanced salt solution), mixed with thoroughly washed mouse myeloma cells (strain P3/Ul) at a ratio of 5:1, and centrifuged at 1,300 rpm for 5 minutes.
The cell pellet was suspended in 1 ml of DMEM(-) medium containing 50% polyethylene glycol 4000 and left to stand for 2 minutes. Subsequently, 10 ml of DMEM(-) medium was slowly added to make a dilution, which was centrifuged at 800 rpm for 5 minutes. The cell pellet was resuspended in 100 ml of a HAT medium containing 20% fetal calf serum and the suspen-sion was distributed in O.l-ml portions among 96 wells in a tissue culture plate. Every 2 - 4 days, half the medium was replaced with a fresh medium. Investigation of antibody titer conducted on the supernatants of the culture after 8 days showed that strong antibody activities were found in 21 of 1,000 wells. Cloning was then performed by a limiting dilution technique so as to select clones that would not show any antibody activity against a mixed plasma of 14 normal subjects or a mixed serum of 14 normal subjects but which would specifically recognize arteriosclerosis related antigens in human sera. As a result, a total of 9 clones of hybridomas were obtained. The final clonins was conducted by a limiting dilution technique so as to isolate a total of 6 clones of hybridomas that were more inactive against normal plasma and sera but which specifically reacted with sera from patients suffering from arteriosclerosis related diseases.
These clones were in~ected intraperitoneally into pristan-treated BALB/c mice and after 10 - 20 days, ascites was colleeted from each animal to obtain monoelonal antibodies.
Of the monoelonal antibodies obtained from the 6 clones of hybridomas, those obtained from cell line 131B
were found to be of immunoglobulin subclass IgG~ by the Ouchterlony method (see Fig. 1) and they had the ability to specifically recognize arteriosclerosis related antigens in human sera. Cell strain 131B has been deposited under the Budapest Treaty with the Fermentation Researeh Institute (FERM) the Ageney of Industrial Seience and Technology, an international depository authority, under Accession Number FERM BP-1676.
(2) Assaying patient sera with monoclonal antibody 131B
The sera to be tested were as follows: serum from a familial hyperlipidemic patient (FH) serum from type III
hyperlipidemic patient (Type III); serum from hypo-beta-lipoproteinemic patient (Hypo ~); serum from patient with myocardial infarction (MI); serum from patient with cerebral infarction (APO); serum from patient with Werner's syndrome (Werner); serum from patient with angina pectoris (Angina);
plasma from normal sub~ect (NP); and a mixed serum prepared from blood samples of 14 normal subjects (NS). Each of these sera was diluted 1,000 folds and 100 ~1 of each dllution was added to an ELISA microplate (Falcon 3912). The plate was left to stand overnight at 4~ to have the homogenate ad-sorbed on it. After treating the microplate with a phosphate buffer solution containing 1% BSA, 100 ~1 of a solution of monoclonal antibody 131B was added and reaction was performed at 37C for 2 hours. After washing the microplate, 100 ~1 of 10,000-fold diluted alkaline phosphatase labelled anti-mouse IgG (Tago) was added and reaction was carried out at 37C for 1 hour. After washing the microplate, 100 ml of 1 mM
diethanolamine buffer solution (pH, 9.8) containing 1 mg/ml of disodium salt of paranitrophenylphosphoric acid and 0.01%
MgCl2 was added and reaction was carried out at 37C for 2 hours. Light absorbance values (OD3 D 5 nm) measured with a microplate colorimeter (Bio-Rad) were used as indices of activity against arteriosclerosis related antigens. The results are shown in Table 1. The monoclonal antibody 131B
of the present invention exhibited high activity levels through highly specific recognition of arteriosclerosis related antigens in FH, Type III, Hypo ~, MI, APO, Werner and Angina. In addition, this monoclonal antibody showed only low activity levels against NP and NS. It was, therefore, clear that monoclonal antibody 131B was capable of specifi-cally recognizing arteriosclerosis related antigens in human sera.
These results are shown graphically in Fig. 2, from which one can see that all sera from hypercholesterolemic patient, Type III hyperlipidemic patient, hypo-beta-lipoproteinemic patient, myocardial infarction patient and cerebral infarction patient showed higher antigenic activity levels than normal plasma and sera.
(3) Arteriosclerosis related antigenic substances in human serum capable of reacting with monoclonal antibody 131B
Sera from familial hypercholesterolemic patients were sub~ected to electrophoresis through an SDS-added polyacryl-amide gel overnight at 40 volts and blotted on a nitro-cellulose membrane. The membrane was thoroughly washed with a phosphate buffer solution containing 0.05% nonionic surfac-tant Tween 20 and thereafter treated overnight with a phos-phate buffer solution containing 5~ 8SA. After washing, the 20 membrane was sub~ected to reaction with monoclonal antibody 131B for 2 hours, then with alkaline phosphatase labelled anti-mouse IgG antibody for 1 hour. After the reaction, the membrane was immersed in a 0.75 M Tris-HCl buffer solution (pH, 8.8) containing 0.2~ paratoluidine salt of 5-bromo-4-chloro-3-indolylphosphoric acid and left to stand overnight at room temperature for immunofluorescent staining. It was estimated from the results of staining that the arterio-sclerosis related antigenic substances in human serum that were specifically recognized by monoclonal antibody 131B had molecular weights of approximately 52,000.
The antigenic substances recognized by monoclonal antibody 131B have specificity for hyperlipidemia and differ from lipoprotein constituents, such as Apo A-I, A-IV, B-100, B-48, E and D, which are said to be closely related to arteriosclerosis (see Fig. 3).
As shown in Fig. 4, the antigenic substances recog-nized by monoclonal antibody 131B were found in fractions that did not float even after 24-hour centrifugation at 30,000 rpm of 1 ml of serum from a familial hyperlipidemic patient that had been ad~usted to a specific gravity of 1.25 with NaBr and thereafter plated with 2 ml of physiological saline having a specific gravity of l.lS3 and 1.6 ml of physiological saline having a specific gravity of 1.063.
Furthermore, the low degree of correlation (R = 0.104) with the cholesterol level of familial hyperlipidemic patients showed that monoclonal antibody 131B does not recognize LDL
or lipoproteins as antigenic substances.
From these results, it is concluded that monoclonal antibody 131B specifically recognizes those antigenic substances which are characteristic of arteriosclerotic diseases and which are commonly found in patient's sera.
It has also been found that this monoclonal antibody is capable of specific recognition of antigenic substances in blood plasma, too.
Table 1 Cholesterol Levels of Sera from Patients Suffering from Arteriosclerosis Related Diseases and Their Antigenic Activities for Monoclonal Antibody 131B
~eas~ ~O~l- Patient No. (mg/dl) ELISA
FH 1 73-02615 91 0.551 FH 2 81-06627 159 0.372 FH . 3 80-48341 219 0.300 FH 4 85-12795 176 0.299 FH 5 78-01909 179 0.223 FH 6 81-42373 223 0.340 FH 7 82-59757 188 0.399 FH 8 82-60373 232 0.376 FH 9 83-11984 223 0.314 FH 10 85-18298 216 0.292 FH 11 81-42373 195 0.259 FH 12 82-57486 225 0.205 FH 13 78-03839 320 0.570 FH 14 78-00694 285 0.434 FH 15 86-26248 245 0.580 FH 16 81-58703 244 0.937 FH 17 86-13848 276 0.650 FH 18 85-46484 250 0.351 FH 19 81-14720 274 0.629 FH 20 78-03537 274 0.591 FH 21 78-04523 241 0.747 FH 22 79-66446 258 0.715 FH 23 79-07111 260 0.832 Table 1 (cont'd) DiseaseSamplePatient No.(mg/dl) ELISA
FH 24 80-19562 241 0.712 FH 25 78-07831 345 0.808 FH 26 83-35843 342 0.561 FH 27 70-01093 236 1.069 FH 28 87-30840 304 0.269 FH 29 80-05283 223 0.681 FH 30 86-18468 229 0.603 FH 31 86-26301 212 0.471 FH 32 85-37071 178 0.446 FH 33 82-29714 215 0.401 FH 34 80-41131 276 0.535 FH 35 80-06511 254 0.211 FH 36 82-45798 211 0.621 FH 37 78-03381 260 0.349 FH 38 87-19069 292 0.546 FH 39 86-33547 205 0.478 FH 40 85-19289 247 0.573 FH 41 87-23854 268 0.724 FH 42 85-04596 227 0.697 FH 43 86-19522 273 0.785 FH 44 79-64322 170 0.475 FH 45 84-29516 226 0.807 FH 46 85-18616 153 0.822 FH 47 81-37201 235 0.549 FH 48 79-36564 202 0.565 able 1 (cont'd) Disease ~Patieot No. (mg/dl) ELISA
FH 49 78-06522 225 0.756 FH 50 78-08511 209 0.291 FH 51 86-07503 199 0.546 FH 52 78-05045 278 0.622 FH 53 86-39926 250 0.586 FH 54 85-32090 162 0.558 FH 55 85-33074 115 0.521 FH 56 81-16704 190 0.727 FH 57 80-27988 251 0.737 FH 58 87-14349 290 0.689 FH 59 80-27368 207 0.818 : FH 60 86-01605 276 0.390 FH 61 84-45499 131 0.951 AP0 62 78-01164 106 0.247 AP0 63 83-18926 123 0.534 AP0 64 78-00900 115 0.939 AP0 65 73-00300 163 0.259 AP0 66 78-09966 148 0.186 AP0 67 78-03785 163 0.360 AP0 68 86-25083 187 0.282 AP0 69 78-00849 199 0.271 AP0 70 78-05165 185 0.296 AP0 7184-10579 205 0.496 AP0 7273-06841 117 0.451 AP0 7378-01373 131 0.302 AP0 7477-02200 170 0.291 Table 1 (cont'd) Di-e~seS~n~l e Patient No. (mg/dl) ELISA
MI 75 84-00571 164 0.335 MI 76 78-00485 161 0.499 MI 77 78-04516 133 0.433 MI 78 78-02674 177 0.423 MI 79 74-03633 178 0.483 MI 80 73-03652 105 0.823 Hypo ~ 81 82-21911 99 0.457 Hypo ~ 82 87-32118 91 0.506 Hypo ~ 83 78-03270 67 0.280 Hypo ~ 84 80-45830 93 0.346 Hypo ~ 85 84-26556 78 0.444 Hypo ~ 86 85-20771 114 0.372 Hypo ~ 87 83-13324 100 0.251 Hypo ~ 88 78-01437 101 0.517 Type III89 79-31979 198 0.545 Type III90 725900 243 0.520 Werner 100 85-40293 183 0.507 Werner 101 81-18040 147 0.447 Anglna 102 83-20213 182 0.205 T.C. : Total cholesterol FH : Familial hyperlipidemia AP0 : Cerebral infarction MI : Myocardial infarction Hypo ~ : Hypo-~-lipoproteinemia Type III : Hyperlipidemia type III
Werner : Werner's syndrome Angina : Angina pectoris Example 2 Arteriosclerotic Wall Recognizing Monoclonal Antibody of Class IgG Prepared Using Arteriosclerotic Lesions as Antigens (1~ Preparation of antigen Human thoracic and abdominal arteriosclerotic walls were extracted and blood vessels were excised in a cool place. Thereafter, the swelling portion of the tunica intima (arteriosclerotic lesion) was peeled off with tweezers and cut into squire pieces (1 mm x 1 mm) with scissors. To these pieces, an aqueous solution (pH, 7.~) containing 1 mM EDTA and 0.1% ethanol was added in portions of 5 - 10 ml per gram on a wet weight basis and a homogenate was prepared with a polytron homogenizer.
A mixture of homogenates originating from 3 patients (equlvalent to 5 specimens) was filtered through 4 superposed sheets of gauze and the filtrate was used as an antigenic solution.
(2) Preparation of monoclonal antibodies To 0.5 mg (200 ~1) of the homogenate, 200 ~1 of Freund's complete ad~avant was added and mixed well. The resulting emulsion was administered subcutaneously into BALB/c mice. After 9 weeks, the same emulsion was admin-istered subcutaneously. After 18 weeks, the final immuniza-tion was conducted by intraperitoneal in;ection of a mixture of 0.5 mg (200 ~1) of the homogenate with 200 ~1 of a DMEM(-) medium. On the ~hird day, spleens were extracted from the sensitized animals. The spleen cells were thoroughly washed with ~MEM(-) medium and mixed with 1 3204hl thoroughly washed mouse myeloma cells (strain P3/Ul) at a ratio of 5:1 and the mixture was centrifuged at 1,000 rpm for 5 minutes.
The cell pellet was suspended in 1 ml of DMEM( - ) medium containing 45% polyethylene glycol 4000 and left to stand for 2 minutes. Subsequently, 10 ml of DMEM( - ) medium was slowly added to make a dilution, which was centrifuged at 1,000 rpm for 5 minutes. The cell pellet was re-suspended in 127 ml of a DMEM medium containing 10% fetal calf serum and the suspension was distributed in l-ml portions among 24 wells in a tissue culture plate.
On the first day of culture, 1 ml of a HAT medium was added and half the medium was replaced with a fresh medium every 1 - 2 days. On the 8th day, the antibody titers of the culture supernatants were examined by the ELISA method.
The previously prepared homogenate of the swelling portion of the tunica intima of human arteriosclerotic lesion was diluted to make a solution having a protein concentration of 10 ~g/ml. A 100-~1 portion of this antigenic solution was added to an ELISA microplate (Nunc), which was left to stand overnight at 4C to have the homogenate adsorbed on it. After treating the microplate with a 2% BSA solution, 100 yl of the culture supernatants from 127 wells was added and reaction was conducted for 2 hours at room temperature. After washing the microplate, 100 ~1 of 6,000-fold diluted alkaline phosphatase labelled anti-mouse IgG antibody (Tago) was added and reaction was carried out for 2 hours at room temperature. After washing the microplate, 100 ~1 of a 1 mM diethanolamine buffer solution (pH, 9.8) containing 1 mg/ml of disodium salt of paranitrophenylphosphoric acid and 0.01% MgCl 2 was added and reaction was carried out for 1 hour at room temperature.
Light absorbance ~OD~os nm) was measured with a microplate colorimeter (Bio-Tech). Sixty-five wells having absorbance values of 0.5 and higher were further examined by autoradiography.
Arteriosclerotic lesions were extracted from arteriosclerotic model rabbits that had been grown with 1% cholesterol-supplemented diets for 4 months, and the extracted lesions were cut into 65 pieces. After treating these pieces with a 5~ BSA solution, 1 ml of the culture supernatants from the 65 wells was added and reaction was 15 performed at 4C for 5 hours. After washing, the reaction mixture was treated with 5~ normal sheep serum and 200 yl (1.7 Ci/ml) of 12 5 I labelled anti-mouse IgG antibody (unlabelled; Tago) was added, followed by reaction at 4C
for 3 hours. After washing, the reaction mixture was 20 subjected to autoradiography with X-ray films (FUJI*Photo Film).
From the 65 wells tested, 10 wells that produced a specific reaction and exhibited high binding efficiencies were selected and subjected to cloning by a limiting dilution technique. As a result, a total of 2 clones of hybridoma was isolated. These hybridomas were injected intraperitoneally into pristan-treated BALB/c mice and a~ter 10 - 16 days,-ascites was collected from each animal to * Trade mark Bl obtain monoclonal antibod,ies. Of the monoclonal antibodies obtained from the 2 clones of hybridomas, those obtained from cell line 125H were found to be immunoglobulin subclass I3G2b by the ELISA method and they had the ability to specifically recognize the swelling portion of the tunica intima of arteriosclerotic wall. Cell line 125H has been deposited under the Budapest Treaty with the FERM, an international depository authority, under Accession Number FERM B~-1675.
(3) Specificity of monoclonal antibody 125H
Using 100 yl of homogenates of the swelling portions of tunica intima of various human arteriosclerotic lesions and those of tunica intima of normal artery (each homogenate being adjusted to a protein content of 10 yg/ml), the reac-tivity of monoclonal antibody 125H was investigated by theELISA method. The results are shown in Table 2 and Fig. 6.
It was established that this monoclonal antibody shows strong activity with human arteriosclerotic lesions.
Measurements of total cholesterol (TC) levels were conducted with a cholesterol assay kit (V-cholestase NISSUI* of Nissui Seiyaku Co., Ltd.) The reactivity of monoclonal antibody 125H with the swelling portion of a rabbit arteriosclerotic tunica intima was also investigated. The results of immunofluorescent staining (by the indirect fluorescent antibody technique) conducted on frozen sections of the arteriosclerotic lesions in arteriosclerotic model rabbits that had been grown with 1% cholesterol-supplemented diets are shown in Fig. 7, and * Trade mark - 23 -the results of staining with Oil-Red O conducted on frozen sections of the same lesions are shown in Fig. 8.
Arteriosclerotic lesions were also extracted from another group of arteriosclerotic model rabbits that had 5 been grown with 1% cholesterol-supplemented diets and the reactivity of monoclonal antibody with these lesions was examined by autoradiography. The results of reaction by indirect autoradiography and the state of the lesions after the reaction are shown in Figs. 9 and 10, respectively.
10 As is clear from these figures, monoclonal antibody 125H
specifically recognized the swelling portion of rabbit arteriosclerotic tunica intima.
On the basis of these results, one may be ~ustified to expect that monoclonal antibody 125H will react with .
arteriosclerotic lesions after administration in vivo. When one wants to prepare a reagent for image analysis to be performed as an ad~unct in the diagnosis of human arterio-sclerosis, he is capable of conducting extensive preliminary tests on dosage, safety and other factors using rabbits before applying said reagent to humans. Therefore, mono-clonal antibody 125H has a potential application in image analyses fo~ the purpose of identifying the site of a specific human arteriosclerotic lesion and examining the extent of its spread.
(4) Antigenic substances in arteriosclerotic wall capable of reacting with monoclonal antibody 125H
Homogenates of swelling tunica intima of arterio-sclerotic lesions and those of a normal arterial wall were subjected to electrophoresis through an SDS-added polyacryl-amide gel for 2 hours at 20 mA, and blotted on a nitro-cellulose membrane. The membrane was thoroughly washed with a phosphate buffer solution containing 0.05~ nonionic surfactant Tween 20 and thereafter treated with a 2~ BSA
solution for 6 hours. After washing, the membrane was subjected to reaction with monoclonal antibody 125H for 16 hours, then with alkaline phosphatase labelled anti-mouse IgG ~ IgM antibody for 2 hours. After the reaction, the 10 membrane was immersed in a 0.75 M Tris-HCl buffer solution (pH, 8.8) containing 0.1% paratoluidine salt of 5-bromo-4-chloro-3-indolylphosphoric acid and left to stand at room temperature for 2 hours. As a result, it was established that the antigenic substances recognized by monoclonal 15 antibody 125H had molecular weights of approximately 44,000 (see Fig. 11).
Table 2 Specificity of Monoclonal Antibody 125H for Arteriosclerotic Lesions - Subject No. Disease Cholesterol/p. (ODcos nm) Rating 1 N740 chest 1 FP 6.7 0.066 2 N740 chest 2FP 10.3 0.020 3 Case 3 abdomen FP 5.7 0.084 4 6320 abdomen 1 FS 3.3 0.099 5 6320 abdomen 2 FP 20.30.036 6 6320 chest 1FS 16.9 0.181 _ 7 S96 chest 2 FP 8.7 0.057 _ 8 S96 abdomen 3 AP 14.60.140 9 N744 chest FP 1.2 0.561 2+
Homogenates of swelling tunica intima of arterio-sclerotic lesions and those of a normal arterial wall were subjected to electrophoresis through an SDS-added polyacryl-amide gel for 2 hours at 20 mA, and blotted on a nitro-cellulose membrane. The membrane was thoroughly washed with a phosphate buffer solution containing 0.05~ nonionic surfactant Tween 20 and thereafter treated with a 2~ BSA
solution for 6 hours. After washing, the membrane was subjected to reaction with monoclonal antibody 125H for 16 hours, then with alkaline phosphatase labelled anti-mouse IgG ~ IgM antibody for 2 hours. After the reaction, the 10 membrane was immersed in a 0.75 M Tris-HCl buffer solution (pH, 8.8) containing 0.1% paratoluidine salt of 5-bromo-4-chloro-3-indolylphosphoric acid and left to stand at room temperature for 2 hours. As a result, it was established that the antigenic substances recognized by monoclonal 15 antibody 125H had molecular weights of approximately 44,000 (see Fig. 11).
Table 2 Specificity of Monoclonal Antibody 125H for Arteriosclerotic Lesions - Subject No. Disease Cholesterol/p. (ODcos nm) Rating 1 N740 chest 1 FP 6.7 0.066 2 N740 chest 2FP 10.3 0.020 3 Case 3 abdomen FP 5.7 0.084 4 6320 abdomen 1 FS 3.3 0.099 5 6320 abdomen 2 FP 20.30.036 6 6320 chest 1FS 16.9 0.181 _ 7 S96 chest 2 FP 8.7 0.057 _ 8 S96 abdomen 3 AP 14.60.140 9 N744 chest FP 1.2 0.561 2+
10 N744 abdomenAP 4.4 0.391 +
11 T0 chest FP 2.8 0.526 2+
12 To abdomen FP 11.3 0.236 +
13 N745 FS 2.1 0.391 +
14 N749-1 C0 3.0 0.340 +
15 N749-2 FP 8.0 0.203 +
16 S102-chest FS 1.7 0.738 3+
17 S102-abdomen FS 1.4 0.554 2+
18 6345 FP 26.6 0.072 19 N751 chest FS 6.8 0.806 4+
20 N751 abdomenFP 8.8 0.348 +
21 6344 chest FP 17.2 0.242 +
22 6344 abdomenFP 23.5 0.157 23 normal 5y chest N 1.0 0.132 24 normal abdomen 0.0 0.163 ~ 32046 1 Lesions FP : fibrous plaque FS : fatty streaks AP : atheromatous plaque CO : calcification N : normal Criteria of rating In terms of ELISA (OD30s nm) values:
>0.8 : 4+
0.6 - 0.8 : 3+
0.4 - 0.6 : 2+
0.2 - 0.4 : +
<0.2 : -Cholesterol/p.
(cholesterol/protein) = (mg/dl)/(mg/ml) Example 3 Monoclonal Antibody of Class IgM Capable of Recognizing Lipid Accumulating Site in the Swelling Portion of Arteriosclerotic Tunica Intima ~1) Preparation of antigen Human thoracic and abdominal arteriosclerotic walls were extracted and blood vessels were excised in a cool place. Thereafter, the swelling portion of the tunica intima (arteriosclerotic lesion) was peeled off with tweezers and cut into square pieces (1 mm x 1 mm) with scissors. To these pieces, an agueous solution (pH, 7.4) containing 1 mM EDTA
and 0.1% ethanol was added in portions of 5 - 10 ml per gram on a wet weight basis and a homogenate was prepared with a polytron homogenizer.
A mixture of homogenates originating from 6 patients (equivalent to 10 specimens) was filtered through 4 superposed sheets of gauze and the filtrate was used as an antigenic solution.
(2) Preparation of monoclonal antibodies To 1 mg (350 yl) of the homogenate, 350 yl of a Freund's complete adjuvant was added and mixed well. The resulting emulsion was administered subcutaneously into BALBtc mice. Following this injection, the same emulsion was administered subcutaneously after 2 and 5 weeks. After 12 weeks, the final immunization was conducted by intra-peritoneal injection of a mixture of 1 mg (350 yl) of thehomogenate with 50 ~1 of a phosphate buffer solution. On the third day, spleens were extracted from the sensitized animals. The spleen cells were thoroughly washed with HHBS
(HEPES buffered Hanks' balanced salt solution) and mixed with thoroughly washed mouse myeloma cells (strain P3/Ul) at a ratio of 8.5:1, and centrifuged at 1,000 rpm for 5 minutes.
The cell pellet was suspended in 1 ml of DMEM(-) medium containing 50% polyethylene glycol 4000 and left to stand for 2 mlnutes. Subsequently, 10 ml of DMEM(-) medium was slowly added to make a dilution, which was centrifuged at 800 rpm for 5 minutes. The cell pellet was resuspended in 35 ml of a HAT medium containing 20% fetal calf serum and the suspension was distributed in 0.1-ml portions among 96 wells ln a tissue culture plate.
Every 2 - 4 days, 50 ~1 of a fresh mediu~ was added.
Investigation of antibody titer conducted on the supernatants of the culture after 13 days showed that strong antibody activities were found in 4 of 344 wells. Cloning was then performed by a limlting dilution technique, whereupon 3 clones of hybridoma were isolated. These clones were in;ected intraperitoneally into pristan-treated BALB/c mice and after 8 - 13 days, ascites was collected from each animal to obtain monoclonal antibodies. Of the monoclonal anti-bodies obtained from the 3 hybridoma clones, those obtained from cell line T256C were found to be of immunoglobulin class IgM by the Ouchterlony method and they had the ability to specifically recognize the lipid accumulating site in the swelling portion of tunica intima of arteriosclerotic wall.
Cell line T256C has been deposited under the Budapest Treaty with the FERM, an international depository authority, under Accession Number FERM BP-1677.
~3) ~abelling of monoclonal antibody T256C with radioisotope Ascites containing monoclonal antibody T256C was salted out with ammonium sulfate and then dialyzed against a 50 mM Tris buffer at pH 8.1. To the dialyzate, trypsin was added in an amount of 2% of the antibody in the presence of cysteine and the mixture was heated at 37C for 4 hours.
Thereafter, the mixture was alkylated with iodoacetamide and sub~ected to gel permeation chromatography on a column of Sephacryl S-200 so as to obtain Fab fractions. Dialysis against a phosphate buffer yielded about 100 yg of Fab.
To this was added about 1 mCi of Nal 2 5I, followed by reaction with 20 yg of chloramine-T for 30 seconds at room temperature. The radioiodination reaction was quenched by addition of 40 yg of sodium metabisulfite and the reaction product was purified by passage through a column of Sephacryl S-300, thereby yielding iodine-labelled monoclonal antibody T256C.
(4) Specificity of monoclonal antibody T256C
Homogenates of the swelling portion of tunica intima of arteriosclerotic lesion and those of tunica intima of normal artery were prepared to have a protein content of 10 ~g/ml, and 100 ~1 of each homogenate was added to an ELISA microplate (Falcon), which was left to stand overnight at 4C to have the homogenate adsorbed on it. After treatlng 10 the microplate with a 1% BSA solution, 100 ~1 of a solution of monoclonal antibody T256C was added and reaction was conducted at room temperature for 2 hours. After washing the microplate, 100 ~1 of 6,000-fold diluted alkaline phosphatase labelled anti-mouse IgG + IgM antibody (Tago) was added and 15 reaction was carried out for 2 hours at room temperature.
After washing the microplate, 100 ~1 of a 1 mM diethanolamine buffer solution (pH, 9.8) containing 1 mg/ml of disodium salt of paranitrophenylphosphoric acid and 0.01% MgCl 2 was added and reaction was carried out for 1 hour at room temperature.
20 Light absorbance (OD~OSnm) was measured with a microplate colorimeter (Bio-Rad). Monoclonal antibody T256C of the present invention was not highly specific for the homogenate of the swelling portion of tunica intima of arteriosclerotic lesion (ODoos nm = 0.072). On the other hand, this antibody was found to have the ability to recognize most specifically the lipid accumulating site in the swelling portion of arteriosclerotic tunica intima. This was established both by immunofluorescent staining (by the indirect fluorescent antibody method) and by staining with Oil-Red O of sections of arteriosclerotic lesion (see Figs. 12 and 13).
Monoclonal antibody T256C was also found to have the ability to cross-react with the swelling portion of rabbit arteriosclerotic tunica intima. The results of immuno-fluorescent staining by the indirect fluorescent antibody method of a section of the arteriosclerotic lesion in an arteriosclerotic model rabbit and the results of staining with Oil-Red O of the same section are shown in Figs. 14 and 15, respectively. As is clear from these figures, monoclonal antibody T256C specifically recognized the lipid accumulating site ln the swelling portion of rabbit arteriosclerotic tunica intima.
On the basis of these results, it may safely be concluded with monoclonal antibody T256C that when one wants to prepare a reagent for image analysis to be performed as an ad~unct in the diagnosis of human arteriosclerosis, he is capable of conducting extensive preliminary tests on dosage, safety and other factors using rabbits before applying said 20 reagent to humans. Therefore, this monoclonal antibody has a potential application in image analyses for the purpose of identifying the site of a specific human arteriosclerotic lesion and examining the extent of its spread.
>0.8 : 4+
0.6 - 0.8 : 3+
0.4 - 0.6 : 2+
0.2 - 0.4 : +
<0.2 : -Cholesterol/p.
(cholesterol/protein) = (mg/dl)/(mg/ml) Example 3 Monoclonal Antibody of Class IgM Capable of Recognizing Lipid Accumulating Site in the Swelling Portion of Arteriosclerotic Tunica Intima ~1) Preparation of antigen Human thoracic and abdominal arteriosclerotic walls were extracted and blood vessels were excised in a cool place. Thereafter, the swelling portion of the tunica intima (arteriosclerotic lesion) was peeled off with tweezers and cut into square pieces (1 mm x 1 mm) with scissors. To these pieces, an agueous solution (pH, 7.4) containing 1 mM EDTA
and 0.1% ethanol was added in portions of 5 - 10 ml per gram on a wet weight basis and a homogenate was prepared with a polytron homogenizer.
A mixture of homogenates originating from 6 patients (equivalent to 10 specimens) was filtered through 4 superposed sheets of gauze and the filtrate was used as an antigenic solution.
(2) Preparation of monoclonal antibodies To 1 mg (350 yl) of the homogenate, 350 yl of a Freund's complete adjuvant was added and mixed well. The resulting emulsion was administered subcutaneously into BALBtc mice. Following this injection, the same emulsion was administered subcutaneously after 2 and 5 weeks. After 12 weeks, the final immunization was conducted by intra-peritoneal injection of a mixture of 1 mg (350 yl) of thehomogenate with 50 ~1 of a phosphate buffer solution. On the third day, spleens were extracted from the sensitized animals. The spleen cells were thoroughly washed with HHBS
(HEPES buffered Hanks' balanced salt solution) and mixed with thoroughly washed mouse myeloma cells (strain P3/Ul) at a ratio of 8.5:1, and centrifuged at 1,000 rpm for 5 minutes.
The cell pellet was suspended in 1 ml of DMEM(-) medium containing 50% polyethylene glycol 4000 and left to stand for 2 mlnutes. Subsequently, 10 ml of DMEM(-) medium was slowly added to make a dilution, which was centrifuged at 800 rpm for 5 minutes. The cell pellet was resuspended in 35 ml of a HAT medium containing 20% fetal calf serum and the suspension was distributed in 0.1-ml portions among 96 wells ln a tissue culture plate.
Every 2 - 4 days, 50 ~1 of a fresh mediu~ was added.
Investigation of antibody titer conducted on the supernatants of the culture after 13 days showed that strong antibody activities were found in 4 of 344 wells. Cloning was then performed by a limlting dilution technique, whereupon 3 clones of hybridoma were isolated. These clones were in;ected intraperitoneally into pristan-treated BALB/c mice and after 8 - 13 days, ascites was collected from each animal to obtain monoclonal antibodies. Of the monoclonal anti-bodies obtained from the 3 hybridoma clones, those obtained from cell line T256C were found to be of immunoglobulin class IgM by the Ouchterlony method and they had the ability to specifically recognize the lipid accumulating site in the swelling portion of tunica intima of arteriosclerotic wall.
Cell line T256C has been deposited under the Budapest Treaty with the FERM, an international depository authority, under Accession Number FERM BP-1677.
~3) ~abelling of monoclonal antibody T256C with radioisotope Ascites containing monoclonal antibody T256C was salted out with ammonium sulfate and then dialyzed against a 50 mM Tris buffer at pH 8.1. To the dialyzate, trypsin was added in an amount of 2% of the antibody in the presence of cysteine and the mixture was heated at 37C for 4 hours.
Thereafter, the mixture was alkylated with iodoacetamide and sub~ected to gel permeation chromatography on a column of Sephacryl S-200 so as to obtain Fab fractions. Dialysis against a phosphate buffer yielded about 100 yg of Fab.
To this was added about 1 mCi of Nal 2 5I, followed by reaction with 20 yg of chloramine-T for 30 seconds at room temperature. The radioiodination reaction was quenched by addition of 40 yg of sodium metabisulfite and the reaction product was purified by passage through a column of Sephacryl S-300, thereby yielding iodine-labelled monoclonal antibody T256C.
(4) Specificity of monoclonal antibody T256C
Homogenates of the swelling portion of tunica intima of arteriosclerotic lesion and those of tunica intima of normal artery were prepared to have a protein content of 10 ~g/ml, and 100 ~1 of each homogenate was added to an ELISA microplate (Falcon), which was left to stand overnight at 4C to have the homogenate adsorbed on it. After treatlng 10 the microplate with a 1% BSA solution, 100 ~1 of a solution of monoclonal antibody T256C was added and reaction was conducted at room temperature for 2 hours. After washing the microplate, 100 ~1 of 6,000-fold diluted alkaline phosphatase labelled anti-mouse IgG + IgM antibody (Tago) was added and 15 reaction was carried out for 2 hours at room temperature.
After washing the microplate, 100 ~1 of a 1 mM diethanolamine buffer solution (pH, 9.8) containing 1 mg/ml of disodium salt of paranitrophenylphosphoric acid and 0.01% MgCl 2 was added and reaction was carried out for 1 hour at room temperature.
20 Light absorbance (OD~OSnm) was measured with a microplate colorimeter (Bio-Rad). Monoclonal antibody T256C of the present invention was not highly specific for the homogenate of the swelling portion of tunica intima of arteriosclerotic lesion (ODoos nm = 0.072). On the other hand, this antibody was found to have the ability to recognize most specifically the lipid accumulating site in the swelling portion of arteriosclerotic tunica intima. This was established both by immunofluorescent staining (by the indirect fluorescent antibody method) and by staining with Oil-Red O of sections of arteriosclerotic lesion (see Figs. 12 and 13).
Monoclonal antibody T256C was also found to have the ability to cross-react with the swelling portion of rabbit arteriosclerotic tunica intima. The results of immuno-fluorescent staining by the indirect fluorescent antibody method of a section of the arteriosclerotic lesion in an arteriosclerotic model rabbit and the results of staining with Oil-Red O of the same section are shown in Figs. 14 and 15, respectively. As is clear from these figures, monoclonal antibody T256C specifically recognized the lipid accumulating site ln the swelling portion of rabbit arteriosclerotic tunica intima.
On the basis of these results, it may safely be concluded with monoclonal antibody T256C that when one wants to prepare a reagent for image analysis to be performed as an ad~unct in the diagnosis of human arteriosclerosis, he is capable of conducting extensive preliminary tests on dosage, safety and other factors using rabbits before applying said 20 reagent to humans. Therefore, this monoclonal antibody has a potential application in image analyses for the purpose of identifying the site of a specific human arteriosclerotic lesion and examining the extent of its spread.
Claims (17)
1. A human arteriosclerosis recognizing monoclonal antibody capable of specifically recognizing a human arteriosclerosis related antigen present in serum from a patient suffering from familial hypercholesterolemia, cerebral infarction or myocardial infarction, said mono-clonal antibody being obtained from a hybridoma produced by fusing anti-human arteriosclerosis antibody producing cells with myeloma cells.
2. A monoclonal antibody according to Claim 1 wherein the human arteriosclerosis related antigen is present in serum from a familial hypercholesterolemic patient.
3. A monoclonal antibody according to Claim 1 wherein the human arteriosclerosis related antigen is present in serum from a patient suffering from cerebral infarction or myocardial infarction.
4. A monoclonal antibody according to Claim 1, which is of immunoglobulin class IgG and is designated as 131B, 125H or T256C.
5. A monoclonal antibody according to Claim 1, which is of immunoglobulin class IgM and is designated as 131B, 125H or T256C.
6. A process for preparing a human arteriosclerosis recognizing monoclonal antibody, which comprises the steps of immunizing a non-human mammal with serum which contains a human arteriosclerosis related antigen and is isolated from a patient suffering from familial hypercholesterolemia, cerabral infarction or myocardial infarction, fusing antibody producing lymphocytes from said mammal with myeloma cells, isolating anti-human arteriosclerosis antibody producing hybridomas, and cultivating said hybridomas.
7. A process according to Claim 6, wherein the non-human mammal is immunized with the serum of a familial hypercholesterolemic patient.
8. A process according to Claim 6, wherein the non-human mammal is immunized with the serum of a patient suffering from cerebral infarction or myocardial infarction.
9. A human arteriosclerosis recognizing monoclonal antibody which is designated as 131B, 125H or T256C and labelled with an enzyme or a radioisotope.
10. A labelled monoclonal antibody according to Claim 9, wherein the radioisotope is 125 I.
11. A reagent for diagnosis of human arterio-sclerosis comprising a human arteriosclerosis recognizing monoclonal antibody which is designated as 131B, 125H or T256C and labelled with an enzyme or a radioisotope.
12. A reagent for imaging diagnosis of human arteriosclerosis comprising a human arteriosclerosis recognizing monoclonal antibody which is designated as 131B, 125H or T256C and labelled with a radioisotope.
13. A diagnostic reagent according to Claim 11, wherein the human arteriosclerosis is myocardial infarction or cerebral infarction.
14. A diagnostic reagent according to claim 12, wherein the human arteriosclerosis is myocardial infarction or cerebral infarction.
15. A diagnostic reagent according to Claim 13 or 14, wherein the radioisotope is 123 I, 125 I or 131 I.
16. A diagnostic reagent according to Claim 13 or 14, wherein the radioisotope is 111 In.
17. A diagnostic reagent according to Claim 13 or 14, wherein the radioisotope is 99mTc.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000558160A CA1320461C (en) | 1988-02-04 | 1988-02-04 | Monoclonal antibody capable of recognizing human arteriosclerosis and process for preparing same |
| ES89103754T ES2050171T3 (en) | 1988-02-04 | 1989-03-03 | MONOCLONAL ANTIBODY ABLE TO RECOGNIZE HUMAN ARTERIOSCLEROSIS AND PROCESS TO PREPARE IT. |
| DE68912165T DE68912165T2 (en) | 1988-02-04 | 1989-03-03 | Monoclonal antibody capable of recognizing an antigen associated with human arteriosclerosis and process for its preparation. |
| EP89103754A EP0334076B1 (en) | 1988-02-04 | 1989-03-03 | Monoclonal antibody capable of recognizing an antigen associated with human arteriosclerosis, and process for preparing the same |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000558160A CA1320461C (en) | 1988-02-04 | 1988-02-04 | Monoclonal antibody capable of recognizing human arteriosclerosis and process for preparing same |
| JP63050162A JP2818658B2 (en) | 1988-03-03 | 1988-03-03 | Monoclonal antibody recognizing human atherosclerosis and method for producing the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1320461C true CA1320461C (en) | 1993-07-20 |
Family
ID=25671698
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000558160A Expired - Fee Related CA1320461C (en) | 1988-02-04 | 1988-02-04 | Monoclonal antibody capable of recognizing human arteriosclerosis and process for preparing same |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0334076B1 (en) |
| CA (1) | CA1320461C (en) |
| DE (1) | DE68912165T2 (en) |
| ES (1) | ES2050171T3 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0485476A4 (en) * | 1989-07-31 | 1993-03-03 | Vasocor | Atherosclerotic plaque specific antigens, antibodies thereto, and uses thereof |
| US5196324A (en) * | 1989-12-15 | 1993-03-23 | Eli Lilly And Company | Monoclonal antibodies reactive with a human atheroma associated antigen |
| US8410251B2 (en) | 2008-06-20 | 2013-04-02 | National University Corporation Okayama University | Antibody against calcified globule and use of the same |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4343734A (en) * | 1980-01-10 | 1982-08-10 | The Children's Hospital Medical Center | Protein diagnostic for atherosclerosis |
| JPS61130238A (en) * | 1984-11-30 | 1986-06-18 | Res Dev Corp Of Japan | Monoclonal antibody for recognizing arteriosclerosis focus and reagent |
| US4745060A (en) * | 1984-12-28 | 1988-05-17 | Board Of Regents, The University Of Texas System | Methods and compositions for the detection of Familial Hypercholesterolemia |
-
1988
- 1988-02-04 CA CA000558160A patent/CA1320461C/en not_active Expired - Fee Related
-
1989
- 1989-03-03 DE DE68912165T patent/DE68912165T2/en not_active Expired - Fee Related
- 1989-03-03 EP EP89103754A patent/EP0334076B1/en not_active Expired - Lifetime
- 1989-03-03 ES ES89103754T patent/ES2050171T3/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| EP0334076B1 (en) | 1994-01-12 |
| DE68912165T2 (en) | 1994-07-21 |
| EP0334076A2 (en) | 1989-09-27 |
| EP0334076A3 (en) | 1990-09-12 |
| ES2050171T3 (en) | 1994-05-16 |
| DE68912165D1 (en) | 1994-02-24 |
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